Human-derived bradeion proteins, DNA coding for the proteins, and uses thereof

ABSTRACT

A human-derived bradeion protein, which has the following properties: (i) it is a transmembranous protein; (ii) it has a structure characteristic of growth hormone and cytokine receptors even in a structure of its transmembranous portion when its structure is determined by a hydrophobicity analysis according to Kyte-Doolittle method; (iii) it is expressed at a high level in a human adult brain, and in less amount in the heart, while it is not expressed in other adult organs or fetus; (iv) it induces programmed cell death (apoptosis) when over-expressed in a cultured human nerve cell lines; (v) it induces termination of cell division and aging when over-expressed in a cultured human normal cell; (vi) it is located in cytoplasm, and forms an intracellular aggregate when overexpressed; and (vii) besides human adult neurons, it is specifically expressed in a human colorectal cancer cell line or in a skin cancer cell line, or an analogue thereof.

FIELD OF THE INVENTION

The present invention relates to a protein involved in long-termsurvival of cranial nerve cell, to DNA encoding the protein, and to usesthereof. More particularly, the present invention relates tohuman-derived bradeion protein or derivatives thereof, to DNA encodingthe protein or the derivatives thereof, to a vector containing the DNA,to a host cell transformed or transfected with the vector, to anantibody immunologically reactive with the protein or the derivativesthereof, and to uses of the DNA or the antibody for detecting a cancer.

BACKGROUND OF THE INVENTION

Cranial nerve cells (neurons) are main elements for controlling survivalof higher order animals. Once the neurons are produced, they do notdivide at all and only gradually exfoliate or go through necrosis.Exfoliation of the neurons occurs in the normal state but isparticularly accelerated by genetic diseases, brain ischemia, or statusepilepticus, or under conditions of poor nutrition and low oxygen. Somedisorders of cranial nerves associated with aging (e.g., dementia)result from deficiency of an absolute amount of functional neuronscaused by accumulation of exfoliated neurons. Thus, the monitoring andcontrol of the exfoliation, as well as regeneration of the functions ofneurons, are the most demanding subject to be solved among the agingproblems.

Cranial nerve cells do not divide at all after the induction phase ofdifferentiation in the process of development, and maintain theirfunctions or is accompanied by gradual deterioration of their functionsuntil the end of the life-time of the individual. They are presumed tohave specific division-interrupting and function-maintaining mechanismsalthough these mechanisms have not yet been clarified. In the centralnervous system, there exist numbers of unknown proteins and signalingsubstances, particularly stimulating substances and receptors thereofinvolved in brain-specific signal transduction, but their details areyet unclear.

Numbers of researches have been conducted worldwide on such an importantelement that controls the survival of the cranial nerve cells. However,none of the elements was clarified in the substance or molecule level,and, prior to everything, it was necessary to develop techniques foranalysis. Recently, the group of Dr. Masashi Yanagisawa and hiscolleagues of the University of Texas, Medical Research Center(authorized by the Howard Hughes Foundation) has succeeded in developinga technique for randomly screening neuropeptides and receptors thereofby using cultured cells, and they have found a substance (named orexin)that directly binds to and stimulates the aperitive center in thehypothalamus, and identified functions of the substance's receptor(Cell, 92, 573-585, 1998). However, such a systematic screening ofsubstances is rare, and currently, stimulating factors involved inbrain-specific signal transduction and receptors thereof are not yetfully clarified.

Under such circumstances, the present inventors have now constructed animproved expression gene (cDNA) library, developed a systematicscreening technique, and succeeded in extraction and selection of genesspecific for cranial nerve cells, thereby accomplishing the presentinvention.

Thus, one object of the present invention is to provide a bradeionprotein involved in long-term survival of cranial nerve cells, and DNAcoding for the bradeion protein.

Another object of the present invention is to provide a vectorcontaining the above-mentioned DNA, and a host cell transformed ortransfected with the vector.

Still another object of the present invention is to use the DNA or anantibody to the protein for detecting cancers.

SUMMARY OF THE INVENTION

The present invention provides a human-derived bradeion protein, whichhas the following properties:

(i) it is a transmembranous protein;

(ii) it has a structure characteristic of growth hormone and cytokinereceptors (even in a structure of its transmembranous portion) when itsstructure is determined by a hydrophobicity analysis according toKyte-Doolittle method;

(iii) it is expressed at a high level in the human adult brain, in lessamount in the heart, while it is not expressed in other adult organs orfetus;

(iv) it induces programmed cell death (apoptosis) when over-expressed incultured human cell lines;

(v) it induces termination of cell division and aging whenover-expressed in cultured human normal cells;

(vi) it is located in cytoplasm, and forms an intracellular aggregatewhen overexpressed; and

(vii) besides human adult neurons, it is specifically expressed in ahuman colorectal cancer cell line or in a skin cancer cell line,

or an analogue thereof.

The proteins of the invention include Bradeion α and Bradeion β proteinshaving the amino acid sequences shown in SEQ ID NOS:2 and 4,respectively. These proteins are not the consequence of alternativesplicing, but coded in the adjacent area (chromosome 17q23) of humangenome. In addition to the above-described properties, Bradeion αinduces programmed cell death when DNA coding for Bradeion α or ananalogue thereof is introduced into a cultured cancer cell.

The term “analogue” as used herein refers to a protein that hasproperties substantially equivalent to those of the human-derivedbradeion proteins (for example, at least the properties of (i), (ii),(iii), (vi) and (vii)), or a protein having an amino acid sequence withdeletions, substitutions or additions of at least one amino acid residuein the amino acid sequence shown in SEQ ID NO:2 or 4, or a proteinhaving an amino acid sequence that is substantially the same as thatshown in SEQ ID NO:2 or 4. Preferably, the analogue of the invention hasat least 90%, preferably at least 95%, more preferably at least 97%homology with the amino acid sequence of SEQ ID NO:2 or 4. The analogueof the invention also includes human bradeion proteins modified ormutated in the amino acid level, and bradeion proteins from non-humanmammals having properties substantially equivalent to those from humans.Only Bradeion β (similar to the human-derived Bradeion β) was found, forexample, in a mouse brain and its homology with the human Bradeion β was94%. The analogues of the invention may be obtained through DNArecombinant techniques by artificial modifications (e.g., site-directedmutagenesis), as long as the original bioactivity of the human bradeionis not impaired. The analogues of the invention may or may not contain asugar chain, or they may be chemically modified with an aqueous polymersuch as polyethylene glycol.

Moreover, the present invention provides DNAs comprising nucleotidesequences coding for the above-defined bradeion proteins or analoguesthereof, and fragments of the DNAs.

Specific examples of such DNAs or fragments thereof include: DNAcomprising the sequence of the nucleotides 129-1943 of SEQ ID NO:1(i.e., DNA encoding human Bradeion α); DNA fragments having at least 15,preferably at least 20, more preferably at least 30 consecutivenucleotides derived from the nucleotides 129-1943 of SEQ ID NO:1; DNAcomprising the full-length nucleotide sequence shown in SEQ ID NO:1; DNAcomprising the sequence of the nucleotides 129-1562 of SEQ ID NO:3(i.e., DNA encoding human Bradeion β); DNA fragments having at least 15,preferably at least 20, more preferably at least 30 consecutivenucleotides derived from the nucleotides 129-1562 of SEQ ID NO:3; andDNA comprising the full-length nucleotide sequence shown in SEQ ID NO:3.In addition, DNA that can hybridize with one of the above-mentioned DNAsunder stringent conditions is also encompassed in the present invention.The stringent conditions as mentioned herein refer to such conditionsthat allow hybridization only when there is at least 90% homology,preferably at least 95% homology, more preferably at least 97% homologywith the nucleotide sequence shown in SEQ ID NO:1 (positions 129-1943)or SEQ ID NO:3 (positions 129-1562). Generally, such conditions allowhybridization at a temperature that is lower by about 5° C.-30° C.,preferably by about 10° C.-25° C. than the melting temperature (Tm) ofthe complete hybrid. Stringent conditions that may be used are describedin J. Sambrook et al., Molecular Cloning, A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1989), esp. “Conditionsfor Hybridization of Oligonucleotide Probes”. The DNA and fragmentsthereof according to the present invention can be used not only for theexpression of the bradeion proteins but also as a probe forhybridization or as a primer for PCR.

The present invention further provides vectors containing DNAs codingfor bradeion proteins or analogues thereof, or fragments of the DNAS.The vector usually contains a promoter that is capable of operablyexpressing the DNAs. In addition to the promoter, the vector may containat least one other element such as an origin of replication, anenhancer, a ribosome-binding site, a transcription termination factor(terminator), a selective marker, an RNA splicing site, or apolyadenylation signal.

The present invention further provides a host cell that has beentransformed or transfected with such a vector. The host cell may be aprokaryotic or eukaryotic cell, preferably an eukaryotic cell, morepreferably a mammalian cell such as a human cell line.

The present invention also provides an antibody that is immunologicallyreactive with the above-defined bradeions or analogues thereof. Theantibody is preferably one that can specifically immuno-react with thebradeion proteins or analogues thereof, and is a polyclonal ormonoclonal antibody.

The invention further provides a method for detecting a cancer,comprising detecting the cancer by using the above-defined DNAs orfragments thereof or the above-defined antibodies as tumor markers.Herein, the cancer includes, but is not limited to, a human colorectalcancer and a human skin cancer. The detection can be conducted byhybridization or immunoassay.

This specification includes all or part of the contents as disclosed inthe specification and/or drawings of Japanese Patent Application No.10-325380, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIGS. 1A and 1B are photographs showing the results of a hydropathyanalysis by the Kyte-Doolittle method, giving distributions ofhydrophobic and hydrophilic portions in Bradeion α together withdistributions in IL2, IL3, IL4 and growth hormone receptors forcomparison;

FIGS. 2A-C are photographs taken with a confocal laser microscopeshowing images of labeled cells observed by gene-transfer andover-expression of Bradeion α and Bradeion β genes in cultured NT2neuron (human undifferentiated nerve cell) and HeLa cell. FIG. 2A showsphotographs for NT2 neuron; FIG. 2B shows photographs for NT2 neuron andHeLa; and FIG. 2C shows electron microscopic images of the cells shownin FIG. 2B (18 and 24 hours after the introduction). FIG. 2A showslocations of bradeion genes identified with EGFP (Enhanced GreenFluorescent Protein; CLONTECH Lab., Inc.) (left), locations of bradeiongenes in mitochondria (center), and overlaid images of the left andcenter images (right).

FIG. 3A is a photograph showing expression of equal amounts of Bradeionα and Bradeion β genes in human cancer cell lines, giving the results ofNorthern blot analysis using radiolabeled bradeion genes. Lane 1 showsexpression in polymyelocytic leukemia, HL60; Lane 2 shows expression inHeLaS3; Lane 3 shows expression in chronicmyologenous leukemia, K-562;Lane 4 shows expression in lymphoblastic leukemia, MOLT-4; Lane 5 showsexpression in Burkitt's lymphoma, Raji; Lane 6 shows expression incolorectal adenocarcinoma, SW480 21, 22; Lane 7 shows expression in lungcarcinoma, A549; and Lane 8 shows expression in melanoma, G361. Theresult of Northern blot analysis using a β-actin gene is shownunderneath as a positive control.

FIG. 3B shows cancer-specific expression of bradeion genes in colorectaladenocarcinoma cell (T1 to T10); skin cancer cells (T11 to T13); andnormal cells (N1 to N2). All specimens are from humans. In the figure,“*” refers to the case where both Bradeion α and Bradeion β genes aredetected without gene mutation; “ND” refers to the case where detectionwas impossible due to denaturation of RNA; “Ad (well)” refers to a welldifferentiated adenocarcinoma; “Ad (mod)” refers to a moderatelydifferentiated adenocarcinoma; “Muc” refers to a mucinouscarcinoma; and“MM” refers to malignant melanoma. Dukes' stage is based on the Dukes'classification. Codon 12 of human K-ras gene (whose wild type sequenceis GGT) is indicated if it has been mutated. This mutation isheterozygous.

FIG. 3C is photographs of the results of in situ hybridization ofspecimens from human cancer tissue, showing stained tissues for T13 andT8 (FIG. 3B) (Antisense: positive control, Sense: negative control).

DETAILED DESCRIPTION OF THE INVENTION

cDNA coding for bradeion proteins of the invention may be obtained asfollows.

First, brain tissue is homogenized in a phenol or phenol-chloroformsolution containing guanidine isothiocyanate, and subjected tohigh-speed centrifugation to be separated into an aqueous layer and anorganic layer. Total RNA contained in the aqueous layer is precipitatedand collected by adding isopropanol, or is collected through sucrose orcesium chloride density-gradient centrifugation. The obtained total RNAis subjected to oligo(dT)-cellulose chromatography to purify mRNA (i.e.,poly(A) RNA) therefrom.

Then, cDNA is synthesized from the mRNA in the presence of a reversetranscriptase. The cDNA is provided with suitable restriction sites andinserted into a phage or plasmid vector having the identical restrictionsites. The thus-obtained vector is used to transform or transfect E.colito produce a CDNA library.

Since the cloned cDNA library includes various DNA fragments withinformation other than that of the DNA of interest, it is necessary toselect the DNA of interest. For this purpose, plaque hybridization orcolony hybridization may usually be employed. According to such methods,plaques (in the case of a phage vector) or colonies (in the case of aplasmid vector) formed on agar are transferred to a nitrocellulosemembrane or a nylon membrane. After being treated with an alkalinesolution, they are bound to a radioactive (³²P) or fluorescence labelingDNA probe that is capable of hybridizing with the DNA of interest, andexposed onto an X-ray film, thereby detecting and collecting a plaque orcolony containing the DNA of interest. Alternatively, the obtained setof clones may be exposed to an inducer such as isopropyl1-thio-β-D-galactoside (hereinafter, referred to as “IPTG”) to forciblyexpress proteins. The proteins are then transferred to a nylon membraneor a cellulose membrane, and a specific antibody for the protein ofinterest is used to immunologically select the corresponding clones.

The cDNA of interest collected from the plaques or colonies thatpositively reacts with the probe is sequenced by Maxiam-Gilbert methodor Sanger-Coulson method.

For cloning and sequencing, for example, methods described in Sambrooket al., Molecular Cloning (supra), Ausubel et al., Current Protocols inMolecular Biology, Green Publishing Company Assoc. and John WileyInterscience, NY, 1992, etc. may be used.

Specifically, as will be described later in Examples, cDNA library fromhuman adult brain is constructed and thereafter cDNA coding for thebradeion proteins of the invention are collected from the positiveclones. As the result of the sequencing analysis, two types of bradeiongenes, i.e., Bradeion α and Bradeion β genes, were found which werepresumably produced due to alternative splicing. The nucleotidesequences of these genes are shown in SEQ ID NOS: 1 and 3, respectively,where the coding regions were at the positions 129-1943 and 129-1562,respectively. The Bradeion α and Bradeion β proteins have amino acidsequences shown in SEQ ID NOS:2 and 4, respectively, as identified fromtheir nucleotide sequences. The Bradeion α DNA has 1815 nucleotides andcodes for a protein having 605 amino acids. The Bradeion β DNA has 1434nucleotides and codes for a protein having 478 amino acids. Met at eachposition 1 of the amino acid sequences of SEQ ID NOS:2 and 4 may bepresent or absent. When these nucleotide and amino acid sequences werecompared to all sequences deposited with the GenBank, it was found thatthe Bradeion α and Bradeion β genes and proteins were novel.

Bradeion α and Bradeion β proteins were found to have a structurecharacteristic of an interleukin receptor even in a structure of itstransmembranous portion, when subjected to the hydropathy analysisaccording to Kyte-Doolittle method (J. Mol. Biol., 157 (1): 105-132,1982). Thus, it has the structure of a transmembrane-type receptor thatis presumably involved in the intracellular signaling mechanism.Bradeion α and Bradeion β proteins are also similar to the relationshipof the tumor suppression genes p53/p73 in that there are two types ofexpression modes, i.e., α- and β-types, and in that either of them isprevalent in various organisms. Formation of an intracellular aggregateis very similar to that seen in human nerve retroplasia caused by atriplet repeat gene expressed substance (Igarashi et al., NatureGenetics, 111-117, 1998; Martindale et al., Nature Genetics, 150-154,1998). Accordingly, it is assumed that the Bradeion α and Bradeion βproteins are greatly associated with specific termination of thedivision of human nerve cells and/or with maintenance of the function ofthe nerve cells after development/differentiation in the normal geneexpression state.

The human-derived bradeion proteins or analogues thereof of theinvention may be obtained, for example, by using gene recombinanttechniques as follows.

Taking account of degeneracy of the genetic codes, a hybridization probehaving at least 15, preferably about 20 to about 50 consecutivenucleotides is constructed based on the nucleotide sequence shown in SEQID NO:1 or 3 or the amino acid sequence shown in SEQ ID NO:2 or 4. Byusing this hybridization probe, DNAs coding for the bradeion proteinsare screened from a genomic DNA library or cDNA library derived fromhuman or non-human mammal brain tissue. The library may be produced byusing commercially available vector such as λ ZAPII or pBluescript®cloning vector (Stratagene Cloning Systems). The plaque or colonycontaining the DNA of interest is selected through plaque hybridizationor colony hybridization.

Alternatively, a DNA sequence generally having 15 to 100 consecutivenucleotides complementary to the nucleotides 129-1943 of SEQ ID NO:1 orthe nucleotides 129-1562 of SEQ ID NO:3, is produced as a primer. Thisprimer can be used to conduct polymerase chain reaction (PCR) in thegenomic DNA library or cDNA library derived from human or non-humanmammalian brain tissue, thereby specifically amplifying the DNA ofinterest. PCR can be conducted through at least 20 cycles, preferably atleast 30 cycles of: denaturation at 94° C. for 1 min.; annealing at 57°C. for 2 min.; and elongation at 70° C. for 3 min. For the PCR, see thetechniques described in Protein Nucleic acid and Enzyme, “Frontier ofPCR method—Basic to Applied Techniques” vol. 4(5), April, 1996Supplement, Kyoritsu Shuppan, Tokyo, Japan.

The cloned or amplified DNA of interest is collected and introduced intoan available suitable expression vector. The obtained vector is used totransform a suitable host cell, which is thereafter cultured in a propermedium for expression of the DNA, to isolate and purify the protein ofinterest.

Examples of the analogue of the present invention include: a proteinhaving a substantially equivalent properties to the human-derivedbradeion (especially, at least the above-mentioned properties of (i),(ii), (iii), (vi) and (vii)); or a protein having an amino acid sequencewith deletions, substitutions or additions of at least one amino acidresidue in the amino acid sequence shown in SEQ ID NO:2 or 4; or aprotein having an amino acid sequence that is substantially the same asthat shown in SEQ ID NO:2 or 4. Preferably, the analogue has at least90%, preferably at least 95%, more preferably at least 97% homology withthe amino acid sequence shown in SEQ ID NO:2 or 4. This means that theamino acid sequence of the analogue may include a modification chosenfrom deletion, substitution or addition of at least one amino acid aslong as the analogue has properties substantially equivalent to those ofthe human Bradeion α or Bradeion β. The mutation may be introduced intothe amino acid sequence shown in SEQ ID NO:2 or 4 through geneticengineering, for example, by well-known techniques such as site-directedmutagenesis/PCR method (S. N. Ho et al., Gene, 77, 51, 1989); andmethods described in Kaoru Saigo and Yumiko Sano (trans.), CurrentProtocols compact version, Experimental Protocols in Molecular BiologyI, June 1997, Maruzen, Tokyo, Japan). Examples of the substitutioninclude substitutions between hydrophobic amino acids (Ala, Val, Leu,Ile, etc.), Ser and Thr, Asp and Glu, Asn and Gln, Lys and Arg, and Pheand Tyr. Examples of the addition include an addition of Met to theN-terminus of a mature protein as seen in products expressed in abacterial host, and an addition of His-tag, or Met-Lys or Met-Argsequence to the N-terminus for facilitating induction of a matureprotein. It is also possible to truncate some amino acid residues at thecarboxyl or amino terminus of the bradeion protein to an extent wherebioactivity is not impaired.

Proteins without sugar chains are produced when a prokaryotic cell suchas bacterium is used as the host cell for expressing DNAs coding for thebradeion proteins or analogues thereof through genetic engineering. Onthe other hand, products with sugar chains may be produced when the DNAsare expressed in an eurokaryotic cell such as fungus, yeast, insect,plant, or mammalian cell. For example, sugar chains may be formed byintroducing an N-binding sugar chain site (generally, Asn-Xaa-Thr/Serwhere Xaa is any amino acid other than Pro) into the sequence. In anyevent, the obtained analogue should have bioactivity substantiallyequivalent to the human bradeions.

Amino groups at the N-terminus or ε-amino groups of lysine of the humanbradeion proteins or analogues thereof of the invention may bechemically bound to an aqueous polymer such as polyethylene glycol(PEG). Pegylation is generally known to reduce or suppress antigenicityor immunogenisity upon in vivo administration of a pegylated product.

The present invention further provides an expression vector containingthe above-described DNA, and a host cell transformed or transfected withthe vector.

The vector of the invention may be in the form of, for example, plasmid,phage, or virus. Other types of vectors may also be used as long as theyare replicable in a host cell. For example, bacterium plasmids (e.g.,pBR322, pKC30, pCFM536, etc.), phage DNAs (e.g., λ phage, etc.), yeastplasmids (e.g., pG-1, etc.), or viral DNAs for mammal host cells (e.g.,baculovirus, vaccinia virus, adenovirus, SV40 and its derivatives, etc.)may be used.

The vector usually contains a replication origin, a selective marker, apromoter, and, if necessary, may contain an enhancer, a transcriptiontermination sequence (terminator), a ribosome-binding site, apolyadenylation signal, etc.

Where the vector is used for E.coli, the replication origin is derivedfrom ColE1, R factor, or F factor. Where the vector is used for yeast,the replication origin is derived from, for example, 2 μm DNA or ARS1.Where the vector is used for a mammalian cell, the replication origin isderived from, for example, SV40, adenovirus, or bovine papilloma virus.

The promoter is a regulator sequence for directing a synthesis of mRNAcoding for the DNA of the invention. Representative examples of thepromoter include adenovirus or SV40 promoter, E.coli lac or trppromoter, phage λP_(L) promoter, ADH, PHO5, GPD, PGK or AOX1 promoter(for yeast), and a promoter derived from nuclear polyhedrosis virus (forBombyx mori cell).

The selective marker is a gene for providing a phenotype to the host inorder to select transformed host cells. Exemplary selective markersinclude kanamycin-resistant gene, ampicillin-resistant gene,tetracycline-resistant gene, and the like (when the vector is used forE.coli); Leu2, Trp1, Ura3 genes, and the like (when the vector is usedfor yeasts); and neomycin-resistant gene, thymidine kynase gene,dihydrofolate reductase gene, and the like (when the vector is used formammalian cells).

Commercially available vectors may be used such as pQE70, pQE60, pQE-9(Qiagen), pBluescript II KS, ptrc99a, pKK223-3, pDR540, pRIT2T(Pharmacia), and pET-11a (Novagen) as bacterium vectors; and pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG and pSVL SV40 (Pharmacia) as eukaryotevectors.

The DNA of the invention may be introduced into the vector by any means.The vector preferably contains a polylinker with various restrictionsites, or a unique restriction site. The DNA of the invention isinserted into a particular restriction site(s) of the vector where ithas been cleaved with a particular restriction endonuclease(s).

The expression vector containing the DNA of the invention with aregulatory sequence is used to transform or transfect a suitable hostcell, thereby expressing and producing the human bradeion protein of theinvention or an analogue thereof in the host cell.

The host cell is, for example, a bacterial cell (e.g., E.coli,streptomyces, or Bacillus subtilis), an eukaryotic cell (e.g.,Aspergillus strain), an yeast cell (e.g., Saccharomyces cerevisiae, ormethanol-assimilating yeast), an insect cell (e.g., Drosophila S2 orSpodoptera Sf9), and a mammalian cell including cultured human cell(e.g., CHO, COS, BHK, 3T3, or C127).

Transformation or tranfection may be conducted by a known method such ascalcium chloride/rubidium chloride method, calcium phosphate method,DEAE-dextran-mediated transfection, or electroporation.

The human-derived bradeion or an analogue thereof of the invention canbe obtained by culturing the host cells which have been transformed ortransfected as described above under the control of the promoter, and bycollecting the produced protein of interest. The host cell is amplifiedor grown to a proper cell density. Then, the promoter is induced byshifting the temperature or by chemical induction (with IPTG, etc.). Thecell is further cultured for a predetermined period. Where the proteinof interest is secreted extracellularly, it can directly be obtainedfrom the medium. Where the protein of interest is presentintracellularly, the cell can be disrupted by physical means (e.g.,sonication or mechanical disruption) or by chemical means (e.g.,lyzozyme or cytolytic agent). Then the protein of interest is purified.The protein may partially or completely be purified from the culturemedium containing the recombinant cells or an extracted solutionthereof, by using routine techniques such as ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,hydrophobic interaction chromatography, affinity chromatography, gelfiltration chromatography, HPLC, electrophoresis, and chromatofocusing,alone or in combination.

The DNA of the invention was gene-transferred into several human celllines or cancer cell lines to examine the functions of the bradeionproteins. As a result, in addition to the above-described findings, thefollowing facts were found out:

(1) when the DNA of the invention is gene-transferred into NT2 neuron(Stratagene), which is a cultured human undifferentiated nerve cell,using Superfect reagent (Qiagen) and over-expressed, the cell death isinduced within 18-24 hours.

(2) when the DNA is similarly over-expressed in cultured human normalcells, termination of the aging and cell division is induced.

(3) when the DNA is gene-transferred into a cultured cancer cell,Bradeion α and Bradeion β genes induce the programmed cell death.

(4) in the course of the induced cell death, the bradeion protein ispresent in cytoplasm, and forms an intracellular aggregate.

As a result of studies using cultured human cancer cell lines, theexpression of bradeion was detected to be specific for colorectal cancerand malignant melanoma (skin cancer), suggesting that it has strongpreferability for tissues and cell types in which bradeion genes areexpressed.

Based on the above-described findings, the bradeion proteins of theinvention seem to allow survival of cranial nerve cells of the centralnervous system in non-dividing state via neuro-stimulating transmission.It seems to be important that over-expression of the bradeion genescontrols cell fate: apoptosis or carcinogenesis, and that normally theexpression ratio of the α-type to the β-type is maintained (at a ratioof 10:1 in a normal cranial nerve cell) . It was also suggested thatdepending on changes in the expression ratio (e.g., 1:1), it may inducedevelopment of cancer. Accordingly, the bradeion proteins are presumedto play an important role in controlling cell fate (termination,apoptosis, and/or carcinogenesis), and also to determine long-termsurvival of cranial nerve cells in non-dividing state in human adultcentral nervous system. Thus, the bradeion proteins are useful formonitoring exfoliated nerve cells associated with aging, studyingnecrosis of nerve cells that occurs during brain ischemia and statusepilepticus, and understanding the survival mechanism of neurons andpathology of brain. The proteins are also useful for producing novelmedicines for treating genetic diseases, and may be applied to geneticdiagnosis and gene therapy of cancers.

Thus, the proteins of the invention or fragments thereof, antibodies tothem, or the DNAs of the invention or fragments thereof may be used fordetecting or diagnosing cancers (e.g., human colorectal carcinoma andhuman skin cancer), for determining the cell fate, and as a targetingmolecule in colorectal cancer and malignant melanoma.

For the above purposes, a polyclonal or monoclonal antibody that isspecific to the proteins of the invention is useful. The antibody,preferably the monoclonal antibody, may be used for diagnosis,vaccination and drug delivery systems.

Such antibodies may be produced, for example, by methods described inSuguru Matsubasi et al., Biochemical Experimental Methods 15,Introduction to Immunological Experiments, 1982, Japan ScientificSocieties Press, Tokyo, Japan; Tatsuo Iwasaki et al., Monoclonalantibody-Hybridoma and ELISA, 1987, Kodansha Scientific, Tokyo, Japan.

The polyclonal antibody can be obtained as follows. First, an antigensolution containing the protein of the invention or a fragment thereofas an antigen is mixed with complete Freund's adjuvant to form anemulsion. The emulsion is then subcutaneously injected into a mammalsuch as rabbit, mouse, goat, bovine or equine. After about 2 weeks, anantigen solution emulsified in incomplete Freund's adjuvant is similarlyinjected into the animal, which is then boosted if necessary, and bloodis drawn from the animal to collect anti-serum as the polyclonalantibody. The anti-serum is further subjected to ammonium sulfateprecipitation or ion exchange chromatography using DEAE cellulose toobtain an IgG fraction. The IgG is subjected to an affinitychromatography on CNBr-activated Sephadex or Sepharose bound to theprotein of the invention or a fragment thereof to mono-specificallypurify the antibody of interest that is bound through immunologicreaction.

The monoclonal antibody can be obtained as follows. First, anantigen-adjuvant emulsion is prepared as described above, which is thenintraperitoneally injected into a mouse (e.g., BALB/c) for immunization.The spleen was removed from the mouse to collect spleen cells, which arethen fused with a myeloma cell (e.g., X63 or NS-1) in the presence ofpolyethylene glycol (e.g., PEG 400). Then, an antibody-producinghybridoma is selected in an HAT medium to obtain the monoclonal antibodyof interest by a cloning method or alternatively from ascites obtainedfrom the mouse after the intraperitoneal injection. Humanized monoclonalantibody may also be prepared by using known techniques as described inTeng et al., Proc. Natl. Acad. Sci. USA, 1983, 80:7308-7312, and Kozboret al., Immunology Today, 1983, 4 (3): 72-79.

The above-described antibody may be used for diagnosing, for example,cancers in a standard immunoassay such as an enzymatic immunoassay, aradioimmunoassay or a fluorescent antibody method.

Where the DNA of the invention or a fragment thereof is used as a probeor a primer, at least 15, preferably at least 20, more preferably atleast 30 consecutive nucleotides derived from the nucleotide sequence ofSEQ ID NO:1 (the nucleotides 129-1943) or SEQ ID NO:3 (the nucleotides129-1562) can be prepared using an automatic DNA synthesizer. Theobtained nucleotide fragment is labeled with an isotope, a fluorescentsubstance or the like, thereby preparing a probe or a PCR primer fordiagnosis. The conditions employed for hybridization are, for example,described in Sambrook et al., Molecular Cloning (supra), and F. M.Ausubel et al., Short Protocols In Molecular Biology, Third Edition,John Wiley & Sons.

EXAMPLES

Hereinafter, the present invention will be described by way of examples.The present invention, however, is not intended to be limited to theseexamples.

Example 1 Cloning and Sequencing of cDNA Coding for Human Bradeion

First, a cDNA library from a human adult brain was constructed using theplasmid vector pCMV SPORT1 (Life Technologies, Inc., USA) which iscapable of linking with a CMV promoter for expressing in an eukaryoticcell. The adult brain was obtained from a 36-year-old white CaucasianAmerican female, and mRNA (poly(A) RNA) was extracted therefrom withTRIzol® reagent (Life Technologies, Inc.) and purified withMESSAGEMAKER® reagent (Life Technologies, Inc.). Then double strandedcDNA synthesis and library construction were initiated by SuperScriptplasmid system.

The prepared mRNA (poly(A) RNA) was linked with NotI primer adapter atits 3′-terminus. Then, a double stranded cDNA was synthesized accordingto a standard method using Superscript II reverse transcriptase and T4DNA polymerase. The 5′-terminus of the CDNA was linked with SalI adapterand 3′-terminus was treated with NotI restriction enzyme so that theCDNA fragment had restriction sites Sal1 and NotI at each end. The cDNAwas separated in sizes by gel filtration chromatography to select andfractionate cDNA having a size of 1 kb or more. The obtained set of cDNAfragments was inserted, by a standard method, into the plasmid vectorPCMV SPORT1 that also had been cleaved with Sal1 and NotI, therebyproducing circular plasmids. These plasmids were introduced into E.coliDH12S cells (Life Technologies, Inc.) by an electroporation method andamplified to construct a library.

The resulting E. coli strains were grown on ampicillin-containing LBagar medium to form colonies. Biodyne A nylon membrane (Pall Corp., US)treated with 10 mM IPTG was placed in close contact with the coloniesand left at 37° C. for 2 hours. The nylon membrane was reacted with theantibody CE5 that specifically recognizes cranial nerve cells [Nature,296, 34-38, 1982]. Positive clones were selected using picoBlue®Immunoscreening Kit (Stratagene, US).

Plasmid DNA was collected from the obtained positive clones to be usedas a ³²P-labeled probe, which was hybridized with nylon membranes withmRNAs specific for different human organs (MTN blot, CLONTECH Lab.,Inc.) to test whether the probe was specific for the brain. Thenucleotide sequences of the cDNAs were determined by sequencing analysisand compared with homologous sequences deposited with the GenBank. Onlythe one that was completely novel was deposited with the GenBank as asequence of interest. The determined nucleotide sequence of the Bradeionα cDNA is shown in SEQ ID NO:1 (GenBank Accession No. AB002110). Thecoding region was at the positions 129-1943. The amino acid sequence ofthe Bradeion α determined based on this nucleotide sequence is shown inSEQ ID NO:2. DNA containing Bradeion α cDNA was deposited with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology (Higashi 1-1-3, Tsukuba-shi,Ibaraki-ken 305-8566, Japan) on Jul. 14, 1998 as FERM P-16897. (Thisdeposit was thereafter transferred to an international deposition underthe terms of Budapest Treaty on Oct. 19, 1999 and was given an AccessionNo. FERM BP-6922.)

Based on the above-described sequence of Bradeion α CDNA, 5′-terminalprimers (described below) were synthesized to systematically screenrelevant genes. For this purpose, Gene Trapper Positive Selection system(Life Technologies, Inc.) was used to screen the above-described genelibrary with the synthesized oligonucleotides and magnet beads. Thesequences of the oligonucleotides used were:

5′-ctgagcaagttcgtgaaggatttc-3′ (SEQ ID NO:5); and

5′-cagtcctctgacaaccagcagta-3′ (SEQ ID NO:6)

As a result, a gene was detected which was named Bradeion β and whosenucleotide sequence is shown in SEQ ID NO:3 (GenBank Accession No.AB008753). The coding region was at the positions 129-1562. The aminoacid sequence of Bradeion β determined based on this nucleotide sequenceis shown in SEQ ID NO:4.

Example 2 Characterization of Bradeion α and Bradeion β Proteins

(1) Hydropathy Analysis

The amino acid sequences of Bradeion α and β proteins determined inExample 1 were subjected to hydropathy analysis by Kyte-Doolittle method(Kyte, J. and Doolittle, R. F. J., J. Mol. Biol., 1982, 157 (1):105-132). This analysis is one method for predicting a high-orderstructure of a protein based on its amino acid sequence, whereby thedistributions of hydrophobic and hydrophilic portions of the protein canbe determined. This analysis therefore allows to study the presence of athree-dimensional structure or a transmembranous domain. FIGS. 1A and 1Bshow the results of the analysis for the bradeion proteins as well asthe results for human-derived IL (interleukin) 2, IL3 and IL4 receptorsand growth hormone receptor for comparison. Referring to the figures,the proteins having the sequences of growth hormone and cytokinereceptors may be divided roughly into three sections, i.e., an assemblyof hydrophobic groups (calculated as positive (“+”) values) as atransmembranous portion (third column from the left, shown in red), anextracellular portion (second column from the left, shown in blue)preceding the transmembranous portion, and a cytoplasmic portion (fourthcolumn from the left, shown in green) following the transmembranousportion. This structure is common among all of the receptors includingthe bradeion proteins of the invention. However, bradeion proteins didnot have a hydrophobic signal peptide (first column from the left, shownin yellow).

As a result, the bradeion proteins of the invention were found to bemembrane proteins with a structure characteristic of an interleukinreceptor even in the structure of a transmembranous portion in the aminoacid sequences.

(2) Localization of Bradeion Proteins

Hybridization with the nylon membranes with mRNAs specific for differenthuman organs (MTN blot, CLONTECH Lab., Inc.) indicated a high levelexpression only in the human adult brain, and a low level of expressionin the heart (≦10% of the expression level in the brain). No expressionwas seen in other organs or in human fetus. Both of the α- and β-typeswere expressed in the adult brain. The difference in types was due tothe gene duplication in the adjacent area (17q23) of human chromosome. Ahomologous gene sequence, but only one of the two types of humanbradeions (i.e., Bradeion β) existed in the mouse brain (94% homology).

(3) Experiment of Overexpression of Bradeion α and Bradeion β Genes inCultured Human Cell Lines

Bradeion α and Bradeion β DNAs were individually gene-transferred intoNT2 neuron (Stratagene, US), which is a cultured human undifferentiatednerve cell line, with Superfect reagent (QIAGEN, US) and over-expressed.The results are shown in FIGS. 2A, 2B and 2C.

FIG. 2A shows images of the labeled cell observed 24 hours afteroverexpression of Bradeion α gene (upper panels) and Bradeion β gene(lower panels). The left images show the locations of Bradeion α andBradeion β. The center images show their locations in mitochondria, andthe right images show the overlaid images of the left and center images.All of the images were observed with a confocal laser microscope. As aresult, it was found that the location of Bradeion α was consistent withthe location in mitochondria (Note: yellow color indicates an overlap ofred and green.) while the location of Bradeion β was not consistent withthe location in mitochondria.

Programmed cell death (apoptosis) was induced within 18-24 hours afterthe transfection of the Bradeion α gene. In the course of the apoptosis,Bradeion α formed an intracellular aggregate. FIG. 2B shows cell imagesobserved at predetermined points of time after the gene transfer. Theleft images show the cultured human cell NT2 neuron (Stratagene) and theright images show the human cancer cell line, HeLa. Both cell linesformed intracellular aggregates, resulting in cell death. To confirmthis fact, the cells of FIG. 2B were observed with an electronmicroscope. As shown in FIG. 2C, the presence of apoptosis corpusclesspecific for programmed cell death (apoptosis) was confirmed.

(4) Correlation of Bradeion α and Bradeion β Genes to Cancers

Although the Bradeion α and Bradeion β genes are only expressed in thenormal adult brain and the heart (about 10% of the expression level ofthe brain), their expression was also found in cultured human cancercell lines. The results are shown in FIGS. 3A, 3B and 3C.

FIG. 3A shows the results of Northern blot regarding expression ofBradeion α and Bradeion β genes in different cultured human cancercells. Specific expressions (signals) were found only in Lane 8 (skincancer cell line G361) and Lane 6 (colorectal adenocarcinoma SW480).

Specimens from human patients (i.e., specimens from pathologic tissues)were used for detection of the cancer-specific expression. As shown inFIG. 3B, the specific expression was observed in 10 specimens havingcolorectal adenocarcinoma (T1 to T10; indicated as Ad), and in 3specimens having skin cancer (T11 to T13; indicated as Muc and MM). FIG.3C shows images of stained cancer cells for confirming thecancer-specific expression.

The above results show that the Bradeion α and Bradeion β proteins andthe genes encoding them can be used as tumor-specific markers ofcolorectal cancer.

ADVANTAGEOUS EFFECT OF THE INVENTION

The bradeion proteins of the invention and the DNA encoding them seem toallow survival of cranial nerve cells of the central nervous system innon-dividing state via neuro-stimulating transmission. Over-expressionof the bradeion genes induces apoptosis. Normally, the in vivoexpression ratio of Bradeion α and Bradeion β proteins is maintained (ata ratio of 10:1 in a normal cranial nerve cell). It is also suggestedthat depending on changes in the expression ratio (e.g., 1:1), thedevelopment of a cancer may be induced. Accordingly, the bradeionproteins and the DNA encoding them were presumed to act as acell-lifetime-prolonging, cancer-development-suppressing factor thatdetermines long-term survival of cranial nerve cells in non-dividingstate after the development/differentiation of the cells. Thus, they areuseful for monitoring exfoliated nerve cells associated with the aging,studying the necrosis of nerve cells that occurs during brain ischemiaand status epilepticus, and understanding the survival mechanism of thecentral nerve cells and pathology of brain. They are also useful forproducing novel medicines for treating genetic diseases, and may beapplied to genetic diagnosis and gene therapy of cancers.

All publications (including patent application) cited herein areincorporated herein by reference in their entirety.

The following are information on SEQ ID NOS:1 to 4 described herein:

SEQ ID NO:1 gaaaggagca agccaggaag ccagacaaca acagcatcaa aacaaggctgtttctgtgtg 60 tgaggaactt tgcctgggag ataaaattag acctagagct ttctgacagggagtctgaag 120 cgtgggacat ggaccgttca ctgggatggc aagggaattc tgtccctgaggacaggactg 180 aacctgggat caaccgtttc ctggaggaca ccacggatga tggagaactgagcaagttcg 240 tgaaggattt ctcaggaaat gcgagctgcc acccaccaga ggctaagacctgggcatcca 300 ggccccaagt cccggagcca aggccccagg ccccggacct ctatgatgatgacctggagt 360 tcagaccccc ctcgcggccc cagtcctctg acaaccagca gtacttctgtgccccagccc 420 ctctcagccc atctgccagg ccccgcagcc catgggggga gcttgatccctatgattcct 480 ctgaggtaga gcctccagcc ctgcctttgc ctttcagtgg gctgctgcaggaagaccggg 540 ggcagggagc aggaatgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgtgtgtttgtgt 600 gtgtgtgtat ctgggaccca tttcagtcct gtgtcagccc tagctccaaaatatctgccc 660 ccaagggcac tggaaatttg cagtttcagc aagggcagga ggcccagctggtggcctcag 720 atgggaactc acagaagtct ggcactgctt ttttaaggct ggggcaaaggcctgaaaggg 780 agagaagatt ggcgctgggt gccggggccc ctttggctcc tcaccgtgatgcattctgcc 840 ttcctgtcta ctacgatgac aaggagtatg tgggctttgc aaccctccccaaccaagtcc 900 accgaaagtc cgtgaagaaa ggctttgact ttaccctcat ggtggcaggagagtctggcc 960 tgggcaaatc cacacttgtc aatagcctct tcctcactga tctgtaccgggaccggaaac 1020 ttcttggtgc tgaagaaagg atcatgcaaa ctgtggagat cactaagcatgcagtggaca 1080 tagaaaaaaa aggtgtgagg ctgcggctca ccattgtgga cacaccaagttttggggatg 1140 cagtcaacaa cacagagtgt atgtctgact ggaagcctgt ggcagaatacattgatcagc 1200 agtttgagca gtatttccga gacgagagtg gcctgaaccg aaagaacatccaagacaaca 1260 gggtgcactg ctgcctgtac ttcatctcac ccttcggcca tgggctccggccattggatg 1320 ttgaattcat gaaggccctg catcagcggg tcaacatcgt gcctatcctggctaaggcag 1380 acacactgac acctcccgaa gtggaccaca agaaacgcaa aatccgggaggagattgagc 1440 attttggaat caagatctat caattcccag actgtgactc tgatgaggatgaggacttca 1500 aattgcagga ccaagcccta aaggaaagca tcccatttgc agtaattggcagcaacactg 1560 tagtagaggc cagagggcgg cgagttcggg gtcgactcta cccctggggcatcgtggaag 1620 tggaaaaccc agggcactgc gactttgtga agctgaggac aatgctggtacgtacccaca 1680 tgcaggacct gaaggatgtg acacgggaga cacattatga gaactaccgggcacagtgca 1740 tccagagcat gacccgcctg gtggtgaatg aacggaatcg caagtatgaccagaagccag 1800 gacaaagctg gcagggggag atcccaagcc tagccttggg tgagaccaagccctactttt 1860 gttcttctat aggccctggg ctcaatctaa gcgggtgctg gggtcctcctcgccttatca 1920 acccttttct ccctttagca aactgactcg ggaaagtggt accgacttccccatccctgc 1980 tgtcccacca gggacagatc cagaaactga gaagcttatc ccagagaaagattaggagct 2040 gcggcggata cacgagatac tacaccaaat accaaaacag ataaaggagaactatttact 2100 ggctttcagc cctggatatt taaatctcct cctcttcttc ctgtccatgccggcccctcc 2160 cagcaccagc tctgctcagg ccccttcagc tactgccact tcgccttacatccctgctga 2220 ctgcccagag actcagagga aataaagttt aataaatctg taggtggcttctgg 2274 SEQ ID NO:2 Met Asp Arg Ser Leu Gly Trp Gln Gly Asn Ser ValPro Glu Asp Arg  1               5                  10                  15 Thr Glu ProGly Ile Asn Arg Phe Leu Glu Asp Thr Thr Asp Asp Gly             20                  25                  30 Glu Leu Ser LysPhe Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His         35                  40                  45 Pro Pro Glu Ala LysThr Trp Ala Ser Arg Pro Gln Val Pro Glu Pro     50                  55                  60 Arg Pro Gln Ala Pro AspLeu Tyr Asp Asp Asp Leu Glu Phe Arg Pro 65                  70                  75                  80 Pro SerArg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys Ala Pro                 85                  90                  95 Ala Pro LeuSer Pro Ser Ala Arg Pro Arg Ser Pro Trp Gly Glu Leu            100                 105                 110 Asp Pro Tyr AspSer Ser Glu Val Glu Pro Pro Ala Leu Pro Leu Pro        115                 120                 125 Phe Ser Gly Leu LeuGln Glu Asp Arg Gly Gln Gly Ala Gly Met Cys    130                 135                 140 Val Cys Val Cys Val CysVal Cys Val Cys Val Phe Val Cys Val Cys145         150                 155                         160 Ile TrpAsp Pro Phe Gln Ser Cys Val Ser Pro Ser Ser Lys Ile Ser                165                 170                 175 Ala Pro LysGly Thr Gly Asn Leu Gln Phe Gln Gln Gly Gln Glu Ala            180                 185                 190 Gln Leu Val AlaSer Asp Gly Asn Ser Gln Lys Ser Gly Thr Ala Phe        195                 200                         205 Leu Arg LeuGly Gln Arg Pro Glu Arg Glu Arg Arg Leu Ala Leu Gly    210                 215                 220 Ala Gly Ala Pro Leu AlaPro His Arg Asp Ala Phe Cys Leu Pro Val225                 230                 235                 240 Tyr TyrAsp Asp Lys Glu Tyr Val Gly Phe Ala Thr Leu Pro Asn Gln                245                 250                 255 Val His ArgLys Ser Val Lys Lys Gly Phe Asp Phe Thr Leu Met Val            260                 265                 270 Ala Gly Glu SerGly Leu Gly Lys Ser Thr Leu Val Asn Ser Leu Phe        275                 280                 285 Leu Thr Asp Leu TyrArg Asp Arg Lys Leu Leu Gly Ala Glu Glu Arg    290                 295                 300 Ile Met Gln Thr Val GluIle Thr Lys His Ala Val Asp Ile Glu Lys305                 310                 315                 320 Lys GlyVal Arg Leu Arg Leu Thr Ile Val Asp Thr Pro Ser Phe Gly                325                 330                 335 Asp Ala ValAsn Asn Thr Glu Cys Met Ser Asp Trp Lys Pro Val Ala            340                 345                 350 Glu Tyr Ile AspGln Gln Phe Glu Gln Tyr Phe Arg Asp Glu Ser Gly        355                 360                 365 Leu Asn Arg Lys AsnIle Gln Asp Asn Arg Val His Cys Cys Leu Tyr    370                 375                 380 Phe Ile Ser Pro Phe GlyHis Gly Leu Arg Pro Leu Asp Val Glu Phe385                 390                 395                 400 Met LysAla Leu His Gln Arg Val Asn Ile Val Pro Ile Leu Ala Lys                405                 410                 415 Ala Asp ThrLeu Thr Pro Pro Glu Val Asp His Lys Lys Arg Lys Ile            420                 425                 430 Arg Glu Glu IleGlu His Phe Gly Ile Lys Ile Tyr Gln Phe Pro Asp        435                 440                 445 Cys Asp Ser Asp GluAsp Glu Asp Phe Lys Leu Gln Asp Gln Ala Leu    450                 455                 460 Lys Glu Ser Ile Pro PheAla Val Ile Gly Ser Asn Thr Val Val Glu465                 470                 475                 480 Ala ArgGly Arg Arg Val Arg Gly Arg Leu Tyr Pro Trp Gly Ile Val                485                 490                 495 Glu Val GluAsn Pro Gly His Cys Asp Phe Val Lys Leu Arg Thr Met            500                 505                 510 Leu Val Arg ThrHis Met Gln Asp Leu Lys Asp Val Thr Arg Glu Thr        515                 520                 525 His Tyr Glu Asn TyrArg Ala Gln Cys Ile Gln Ser Met Thr Arg Leu    530                 535                 540 Val Val Asn Glu Arg AsnArg Lys Tyr Asp Gln Lys Pro Gly Gln Ser545                 550                 555                 560 Trp GlnGly Glu Ile Pro Ser Leu Ala Leu Gly Glu Thr Lys Pro Tyr                565                 570                 575 Phe Cys SerSer Ile Gly Pro Gly Leu Asn Leu Ser Gly Cys Trp Gly            580                 585                 590 Pro Pro Arg LeuIle Asn Pro Phe Leu Pro Leu Ala Asn        595                 600                 605 SEQ ID NO:3gaaaggagca agccaggaag ccagacaaca acagcatcaa aacaaggctg tttctgtgtg 60tgaggaactt tgcctgggag ataaaattag acctagagct ttctgacagg gagtctgaag 120cgtgggacat ggaccgttca ctgggatggc aagggaattc tgtccctgag gacaggactg 180aagctgggat caagcgtttc ctggaggaca ccacggatga tggagaactg agcaagttcg 240tgaaggattt ctcaggaaat gcgagctgcc acccaccaga ggctaagacc tgggcatcca 300ggccccaagt cccggagcca aggccccagg ccccggacct ctatgatgat gacctggagt 360tcagaccccc ctcgcggccc cagtcctctg acaaccagca gtacttctgt gccccagccc 420ctctcagccc atctgccagg ccccgcagcc catggggcaa gcttgatccc tatgattcct 480ctgaggatga caaggagtat gtgggctttg caaccctccc caaccaagtc caccgaaagt 540ccgtgaagaa aggctttgac tttaccctca tggtggcagg agagtctggc ctgggcaaat 600ccacacttgt caatagcctc ttcctcactg atctgtaccg ggaccggaaa cttcttggtg 660ctgaagagag gatcatgcaa actgtggaga tcactaagca tgcagtggac atagaagaga 720agggtgtgag gctgcggctc accattgtgg acacaccagg ttttggggat gcagtcaaca 780acacagagtg ctggaagcct gtggcagaat acattgatca gcagtttgag cagtatttcc 840gagacgagag tggcctgaac cgaaagaaca tccaagacaa cagggtgcac tgctgcctgt 900acttcatctc acccttcggc catgggctcc ggccattgga tgttgaattc atgaaggccc 960tgcatcagcg ggtcaacatc gtgcctatcc tggctaaggc agacacactg acacctcccg 1020aagtggacca caagaaacgc aaaatccggg aggagattga gcattttgga atcaagatct 1080atcaattccc agactgtgac tctgatgagg atgaggactt caaattgcag gaccaagccc 1140taaaggaaag catcccattt gcagtaattg gcagcaacac tgtagtagag gccagagggc 1200ggcgagttcg gggtcgactc tacccctggg gcatcgtgga agtggaaaac ccagggcact 1260gcgactttgt gaagctgagg acaatgctgg tacgtaccca catgcaggac ctgaaggatg 1320tgacacggga gacacattat gagaactacc gggcacagtg catccagagc atgacccgcc 1380tggtggtgaa ggaacggaat cgcaacaaac tgactcggga aagtggtacc gacttcccca 1440tccctgctgt cccaccaggg acagatccag aaactgagaa gcttatccga gagaaagatg 1500aggagctgcg gcggatgcag gagatgctac acaaaataca aaaacagatg aaggagaact 1560attaactggc tttcagccct ggatatttaa atctcctcct cttcttcctg tccatgccgg 1620cccctcccag caccagctct gctcaggccc cttcagctac tgccacttcg cctaacatcc 1680ctgctgactg cccagagact cagaggaaat aaagtttaat aaatctgtag gtggc 1735 SEQ IDNO:4 Met Asp Arg Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu Asp Arg  1               5                  10                  15 Thr Glu AlaGly Ile Lys Arg Phe Leu Glu Asp Thr Thr Asp Asp Gly             20                  25                  30 Glu Leu Ser LysPhe Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His         35                  40                  45 Pro Pro Glu Ala LysThr Trp Ala Ser Arg Pro Gln Val Pro Glu Pro     50                  55                  60 Arg Pro Gln Ala Pro AspLeu Tyr Asp Asp Asp Leu Glu Phe Arg Pro  65                 70                  75                   80 Pro SerArg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys Ala Pro                 85                  90                  95 Ala Pro LeuSer Pro Ser Ala Arg Pro Arg Ser Pro Trp Gly Lys Leu            100                 105                 110 Asp Pro Tyr AspSer Ser Glu Asp Asp Lys Glu Tyr Val Gly Phe Ala        115                 120                 125 Thr Leu Pro Asn GlnVal His Arg Lys Ser Val Lys Lys Gly Phe Asp    130                 135                 140 Phe Thr Leu Met Val AlaGly Glu Ser Gly Leu Gly Lys Ser Thr Leu145                 150                 155                 160 Val AsnSer Leu Phe Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu                165                 170                 175 Gly Ala GluGlu Arg Ile Met Gln Thr Val Glu Ile Thr Lys His Ala            180                 185                 190 Val Asp Ile GluGlu Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp        195                 200                 205 Thr Pro Gly Phe GlyAsp Ala Val Asn Asn Thr Glu Cys Val Lys Pro    210                 215                 220 Val Ala Glu Tyr Ile AspGln Gln Phe Glu Gln Tyr Phe Arg Asp Glu225                 230                 235                 240 Ser GlyLeu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val His Cys Cys                245                 250                 255 Leu Tyr PheIle Ser Pro Phe Gly His Gly Leu Arg Pro Leu Asp Val            260                 265                 270 Glu Phe Met LysAla Leu His Gln Arg Val Asn Ile Val Pro Ile Leu        275                 280                 285 Ala Lys Ala Asp ThrLeu Thr Pro Pro Glu Val Asp His Lys Lys Arg    290                 295                 300 Lys Ile Arg Glu Glu IleGlu His Phe Gly Ile Lys Ile Tyr Gln Phe305                 310                 315                 320 Pro AspCys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp Gln                325                 330                 335 Ala Leu LysGlu Ser Ile Pro Phe Ala Val Ile Gly Ser Asn Thr Val            340                 345                 350 Val Glu Ala ArgGly Arg Arg Val Arg Gly Arg Leu Tyr Pro Trp Gly        355                 360                 365 Ile Val Glu Val GluAsn Pro Gly His Cys Asp Phe Val Lys Leu Arg    370                 375                 380 Thr Met Leu Val Arg ThrHis Met Gln Asp Leu Lys Asp Val Thr Arg385                 390                 395                 400 Glu ThrHis Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr                405                 410                 415 Arg Leu ValVal Lys Glu Arg Asn Arg Asn Lys Leu Thr Arg Glu Ser            420                 425                 430 Gly Thr Asp PhePro Ile Pro Ala Val Pro Pro Gly Thr Asp Pro Glu        435                 440                 445 Thr Glu Lys Leu IleArg Glu Lys Asp Glu Glu Leu Arg Arg Met Asp    450                 455                 460 Glu Met Leu His Lys IleGln Lys Gln Met Lys Glu Asn Tyr465                 470                 475

6 1 2274 DNA Homo sapiens CDS (129)..(1943) 1 gaaaggagca agccaggaagccagacaaca acagcatcaa aacaaggctg tttctgtgtg 60 tgaggaactt tgcctgggagataaaattag acctagagct ttctgacagg gagtctgaag 120 cgtgggac atg gac cgt tcactg gga tgg caa ggg aat tct gtc cct gag 170 Met Asp Arg Ser Leu Gly TrpGln Gly Asn Ser Val Pro Glu 1 5 10 gac agg act gaa cct ggg atc aac cgtttc ctg gag gac acc acg gat 218 Asp Arg Thr Glu Pro Gly Ile Asn Arg PheLeu Glu Asp Thr Thr Asp 15 20 25 30 gat gga gaa ctg agc aag ttc gtg aaggat ttc tca gga aat gcg agc 266 Asp Gly Glu Leu Ser Lys Phe Val Lys AspPhe Ser Gly Asn Ala Ser 35 40 45 tgc cac cca cca gag gct aag acc tgg gcatcc agg ccc caa gtc ccg 314 Cys His Pro Pro Glu Ala Lys Thr Trp Ala SerArg Pro Gln Val Pro 50 55 60 gag cca agg ccc cag gcc ccg gac ctc tat gatgat gac ctg gag ttc 362 Glu Pro Arg Pro Gln Ala Pro Asp Leu Tyr Asp AspAsp Leu Glu Phe 65 70 75 aga ccc ccc tcg cgg ccc cag tcc tct gac aac cagcag tac ttc tgt 410 Arg Pro Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln GlnTyr Phe Cys 80 85 90 gcc cca gcc cct ctc agc cca tct gcc agg ccc cgc agccca tgg ggg 458 Ala Pro Ala Pro Leu Ser Pro Ser Ala Arg Pro Arg Ser ProTrp Gly 95 100 105 110 gag ctt gat ccc tat gat tcc tct gag gta gag cctcca gcc ctg cct 506 Glu Leu Asp Pro Tyr Asp Ser Ser Glu Val Glu Pro ProAla Leu Pro 115 120 125 ttg cct ttc agt ggg ctg ctg cag gaa gac cgg gggcag gga gca gga 554 Leu Pro Phe Ser Gly Leu Leu Gln Glu Asp Arg Gly GlnGly Ala Gly 130 135 140 atg tgt gtg tgt gtg tgt gtg tgt gtg tgt gtg tgtgtg ttt gtg tgt 602 Met Cys Val Cys Val Cys Val Cys Val Cys Val Cys ValPhe Val Cys 145 150 155 gtg tgt atc tgg gac cca ttt cag tcc tgt gtc agccct agc tcc aaa 650 Val Cys Ile Trp Asp Pro Phe Gln Ser Cys Val Ser ProSer Ser Lys 160 165 170 ata tct gcc ccc aag ggc act gga aat ttg cag tttcag caa ggg cag 698 Ile Ser Ala Pro Lys Gly Thr Gly Asn Leu Gln Phe GlnGln Gly Gln 175 180 185 190 gag gcc cag ctg gtg gcc tca gat ggg aac tcacag aag tct ggc act 746 Glu Ala Gln Leu Val Ala Ser Asp Gly Asn Ser GlnLys Ser Gly Thr 195 200 205 gct ttt tta agg ctg ggg caa agg cct gaa agggag aga aga ttg gcg 794 Ala Phe Leu Arg Leu Gly Gln Arg Pro Glu Arg GluArg Arg Leu Ala 210 215 220 ctg ggt gcc ggg gcc cct ttg gct cct cac cgtgat gca ttc tgc ctt 842 Leu Gly Ala Gly Ala Pro Leu Ala Pro His Arg AspAla Phe Cys Leu 225 230 235 cct gtc tac tac gat gac aag gag tat gtg ggcttt gca acc ctc ccc 890 Pro Val Tyr Tyr Asp Asp Lys Glu Tyr Val Gly PheAla Thr Leu Pro 240 245 250 aac caa gtc cac cga aag tcc gtg aag aaa ggcttt gac ttt acc ctc 938 Asn Gln Val His Arg Lys Ser Val Lys Lys Gly PheAsp Phe Thr Leu 255 260 265 270 atg gtg gca gga gag tct ggc ctg ggc aaatcc aca ctt gtc aat agc 986 Met Val Ala Gly Glu Ser Gly Leu Gly Lys SerThr Leu Val Asn Ser 275 280 285 ctc ttc ctc act gat ctg tac cgg gac cggaaa ctt ctt ggt gct gaa 1034 Leu Phe Leu Thr Asp Leu Tyr Arg Asp Arg LysLeu Leu Gly Ala Glu 290 295 300 gaa agg atc atg caa act gtg gag atc actaag cat gca gtg gac ata 1082 Glu Arg Ile Met Gln Thr Val Glu Ile Thr LysHis Ala Val Asp Ile 305 310 315 gaa aaa aaa ggt gtg agg ctg cgg ctc accatt gtg gac aca cca agt 1130 Glu Lys Lys Gly Val Arg Leu Arg Leu Thr IleVal Asp Thr Pro Ser 320 325 330 ttt ggg gat gca gtc aac aac aca gag tgtatg tct gac tgg aag cct 1178 Phe Gly Asp Ala Val Asn Asn Thr Glu Cys MetSer Asp Trp Lys Pro 335 340 345 350 gtg gca gaa tac att gat cag cag tttgag cag tat ttc cga gac gag 1226 Val Ala Glu Tyr Ile Asp Gln Gln Phe GluGln Tyr Phe Arg Asp Glu 355 360 365 agt ggc ctg aac cga aag aac atc caagac aac agg gtg cac tgc tgc 1274 Ser Gly Leu Asn Arg Lys Asn Ile Gln AspAsn Arg Val His Cys Cys 370 375 380 ctg tac ttc atc tca ccc ttc ggc catggg ctc cgg cca ttg gat gtt 1322 Leu Tyr Phe Ile Ser Pro Phe Gly His GlyLeu Arg Pro Leu Asp Val 385 390 395 gaa ttc atg aag gcc ctg cat cag cgggtc aac atc gtg cct atc ctg 1370 Glu Phe Met Lys Ala Leu His Gln Arg ValAsn Ile Val Pro Ile Leu 400 405 410 gct aag gca gac aca ctg aca cct cccgaa gtg gac cac aag aaa cgc 1418 Ala Lys Ala Asp Thr Leu Thr Pro Pro GluVal Asp His Lys Lys Arg 415 420 425 430 aaa atc cgg gag gag att gag catttt gga atc aag atc tat caa ttc 1466 Lys Ile Arg Glu Glu Ile Glu His PheGly Ile Lys Ile Tyr Gln Phe 435 440 445 cca gac tgt gac tct gat gag gatgag gac ttc aaa ttg cag gac caa 1514 Pro Asp Cys Asp Ser Asp Glu Asp GluAsp Phe Lys Leu Gln Asp Gln 450 455 460 gcc cta aag gaa agc atc cca tttgca gta att ggc agc aac act gta 1562 Ala Leu Lys Glu Ser Ile Pro Phe AlaVal Ile Gly Ser Asn Thr Val 465 470 475 gta gag gcc aga ggg cgg cga gttcgg ggt cga ctc tac ccc tgg ggc 1610 Val Glu Ala Arg Gly Arg Arg Val ArgGly Arg Leu Tyr Pro Trp Gly 480 485 490 atc gtg gaa gtg gaa aac cca gggcac tgc gac ttt gtg aag ctg agg 1658 Ile Val Glu Val Glu Asn Pro Gly HisCys Asp Phe Val Lys Leu Arg 495 500 505 510 aca atg ctg gta cgt acc cacatg cag gac ctg aag gat gtg aca cgg 1706 Thr Met Leu Val Arg Thr His MetGln Asp Leu Lys Asp Val Thr Arg 515 520 525 gag aca cat tat gag aac taccgg gca cag tgc atc cag agc atg acc 1754 Glu Thr His Tyr Glu Asn Tyr ArgAla Gln Cys Ile Gln Ser Met Thr 530 535 540 cgc ctg gtg gtg aat gaa cggaat cgc aag tat gac cag aag cca gga 1802 Arg Leu Val Val Asn Glu Arg AsnArg Lys Tyr Asp Gln Lys Pro Gly 545 550 555 caa agc tgg cag ggg gag atccca agc cta gcc ttg ggt gag acc aag 1850 Gln Ser Trp Gln Gly Glu Ile ProSer Leu Ala Leu Gly Glu Thr Lys 560 565 570 ccc tac ttt tgt tct tct ataggc cct ggg ctc aat cta agc ggg tgc 1898 Pro Tyr Phe Cys Ser Ser Ile GlyPro Gly Leu Asn Leu Ser Gly Cys 575 580 585 590 tgg ggt cct cct cgc cttatc aac cct ttt ctc cct tta gca aac 1943 Trp Gly Pro Pro Arg Leu Ile AsnPro Phe Leu Pro Leu Ala Asn 595 600 605 tgactcggga aagtggtacc gacttccccatccctgctgt cccaccaggg acagatccag 2003 aaactgagaa gcttatccca gagaaagattaggagctgcg gcggatacac gagatactac 2063 accaaatacc aaaacagata aaggagaactatttactggc tttcagccct ggatatttaa 2123 atctcctcct cttcttcctg tccatgccggcccctcccag caccagctct gctcaggccc 2183 cttcagctac tgccacttcg ccttacatccctgctgactg cccagagact cagaggaaat 2243 aaagtttaat aaatctgtag gtggcttctg g2274 2 605 PRT Homo sapiens 2 Met Asp Arg Ser Leu Gly Trp Gln Gly AsnSer Val Pro Glu Asp Arg 1 5 10 15 Thr Glu Pro Gly Ile Asn Arg Phe LeuGlu Asp Thr Thr Asp Asp Gly 20 25 30 Glu Leu Ser Lys Phe Val Lys Asp PheSer Gly Asn Ala Ser Cys His 35 40 45 Pro Pro Glu Ala Lys Thr Trp Ala SerArg Pro Gln Val Pro Glu Pro 50 55 60 Arg Pro Gln Ala Pro Asp Leu Tyr AspAsp Asp Leu Glu Phe Arg Pro 65 70 75 80 Pro Ser Arg Pro Gln Ser Ser AspAsn Gln Gln Tyr Phe Cys Ala Pro 85 90 95 Ala Pro Leu Ser Pro Ser Ala ArgPro Arg Ser Pro Trp Gly Glu Leu 100 105 110 Asp Pro Tyr Asp Ser Ser GluVal Glu Pro Pro Ala Leu Pro Leu Pro 115 120 125 Phe Ser Gly Leu Leu GlnGlu Asp Arg Gly Gln Gly Ala Gly Met Cys 130 135 140 Val Cys Val Cys ValCys Val Cys Val Cys Val Phe Val Cys Val Cys 145 150 155 160 Ile Trp AspPro Phe Gln Ser Cys Val Ser Pro Ser Ser Lys Ile Ser 165 170 175 Ala ProLys Gly Thr Gly Asn Leu Gln Phe Gln Gln Gly Gln Glu Ala 180 185 190 GlnLeu Val Ala Ser Asp Gly Asn Ser Gln Lys Ser Gly Thr Ala Phe 195 200 205Leu Arg Leu Gly Gln Arg Pro Glu Arg Glu Arg Arg Leu Ala Leu Gly 210 215220 Ala Gly Ala Pro Leu Ala Pro His Arg Asp Ala Phe Cys Leu Pro Val 225230 235 240 Tyr Tyr Asp Asp Lys Glu Tyr Val Gly Phe Ala Thr Leu Pro AsnGln 245 250 255 Val His Arg Lys Ser Val Lys Lys Gly Phe Asp Phe Thr LeuMet Val 260 265 270 Ala Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu Val AsnSer Leu Phe 275 280 285 Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu GlyAla Glu Glu Arg 290 295 300 Ile Met Gln Thr Val Glu Ile Thr Lys His AlaVal Asp Ile Glu Lys 305 310 315 320 Lys Gly Val Arg Leu Arg Leu Thr IleVal Asp Thr Pro Ser Phe Gly 325 330 335 Asp Ala Val Asn Asn Thr Glu CysMet Ser Asp Trp Lys Pro Val Ala 340 345 350 Glu Tyr Ile Asp Gln Gln PheGlu Gln Tyr Phe Arg Asp Glu Ser Gly 355 360 365 Leu Asn Arg Lys Asn IleGln Asp Asn Arg Val His Cys Cys Leu Tyr 370 375 380 Phe Ile Ser Pro PheGly His Gly Leu Arg Pro Leu Asp Val Glu Phe 385 390 395 400 Met Lys AlaLeu His Gln Arg Val Asn Ile Val Pro Ile Leu Ala Lys 405 410 415 Ala AspThr Leu Thr Pro Pro Glu Val Asp His Lys Lys Arg Lys Ile 420 425 430 ArgGlu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln Phe Pro Asp 435 440 445Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp Gln Ala Leu 450 455460 Lys Glu Ser Ile Pro Phe Ala Val Ile Gly Ser Asn Thr Val Val Glu 465470 475 480 Ala Arg Gly Arg Arg Val Arg Gly Arg Leu Tyr Pro Trp Gly IleVal 485 490 495 Glu Val Glu Asn Pro Gly His Cys Asp Phe Val Lys Leu ArgThr Met 500 505 510 Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val ThrArg Glu Thr 515 520 525 His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln SerMet Thr Arg Leu 530 535 540 Val Val Asn Glu Arg Asn Arg Lys Tyr Asp GlnLys Pro Gly Gln Ser 545 550 555 560 Trp Gln Gly Glu Ile Pro Ser Leu AlaLeu Gly Glu Thr Lys Pro Tyr 565 570 575 Phe Cys Ser Ser Ile Gly Pro GlyLeu Asn Leu Ser Gly Cys Trp Gly 580 585 590 Pro Pro Arg Leu Ile Asn ProPhe Leu Pro Leu Ala Asn 595 600 605 3 1735 DNA Homo sapiens CDS(129)..(1562) 3 gaaaggagca agccaggaag ccagacaaca acagcatcaa aacaaggctgtttctgtgtg 60 tgaggaactt tgcctgggag ataaaattag acctagagct ttctgacagggagtctgaag 120 cgtgggac atg gac cgt tca ctg gga tgg caa ggg aat tct gtccct gag 170 Met Asp Arg Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu 1 510 gac agg act gaa gct ggg atc aag cgt ttc ctg gag gac acc acg gat 218Asp Arg Thr Glu Ala Gly Ile Lys Arg Phe Leu Glu Asp Thr Thr Asp 15 20 2530 gat gga gaa ctg agc aag ttc gtg aag gat ttc tca gga aat gcg agc 266Asp Gly Glu Leu Ser Lys Phe Val Lys Asp Phe Ser Gly Asn Ala Ser 35 40 45tgc cac cca cca gag gct aag acc tgg gca tcc agg ccc caa gtc ccg 314 CysHis Pro Pro Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro 50 55 60 gagcca agg ccc cag gcc ccg gac ctc tat gat gat gac ctg gag ttc 362 Glu ProArg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu Glu Phe 65 70 75 aga cccccc tcg cgg ccc cag tcc tct gac aac cag cag tac ttc tgt 410 Arg Pro ProSer Arg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys 80 85 90 gcc cca gcccct ctc agc cca tct gcc agg ccc cgc agc cca tgg ggc 458 Ala Pro Ala ProLeu Ser Pro Ser Ala Arg Pro Arg Ser Pro Trp Gly 95 100 105 110 aag cttgat ccc tat gat tcc tct gag gat gac aag gag tat gtg ggc 506 Lys Leu AspPro Tyr Asp Ser Ser Glu Asp Asp Lys Glu Tyr Val Gly 115 120 125 ttt gcaacc ctc ccc aac caa gtc cac cga aag tcc gtg aag aaa ggc 554 Phe Ala ThrLeu Pro Asn Gln Val His Arg Lys Ser Val Lys Lys Gly 130 135 140 ttt gacttt acc ctc atg gtg gca gga gag tct ggc ctg ggc aaa tcc 602 Phe Asp PheThr Leu Met Val Ala Gly Glu Ser Gly Leu Gly Lys Ser 145 150 155 aca cttgtc aat agc ctc ttc ctc act gat ctg tac cgg gac cgg aaa 650 Thr Leu ValAsn Ser Leu Phe Leu Thr Asp Leu Tyr Arg Asp Arg Lys 160 165 170 ctt cttggt gct gaa gag agg atc atg caa act gtg gag atc act aag 698 Leu Leu GlyAla Glu Glu Arg Ile Met Gln Thr Val Glu Ile Thr Lys 175 180 185 190 catgca gtg gac ata gaa gag aag ggt gtg agg ctg cgg ctc acc att 746 His AlaVal Asp Ile Glu Glu Lys Gly Val Arg Leu Arg Leu Thr Ile 195 200 205 gtggac aca cca ggt ttt ggg gat gca gtc aac aac aca gag tgc tgg 794 Val AspThr Pro Gly Phe Gly Asp Ala Val Asn Asn Thr Glu Cys Trp 210 215 220 aagcct gtg gca gaa tac att gat cag cag ttt gag cag tat ttc cga 842 Lys ProVal Ala Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe Arg 225 230 235 gacgag agt ggc ctg aac cga aag aac atc caa gac aac agg gtg cac 890 Asp GluSer Gly Leu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val His 240 245 250 tgctgc ctg tac ttc atc tca ccc ttc ggc cat ggg ctc cgg cca ttg 938 Cys CysLeu Tyr Phe Ile Ser Pro Phe Gly His Gly Leu Arg Pro Leu 255 260 265 270gat gtt gaa ttc atg aag gcc ctg cat cag cgg gtc aac atc gtg cct 986 AspVal Glu Phe Met Lys Ala Leu His Gln Arg Val Asn Ile Val Pro 275 280 285atc ctg gct aag gca gac aca ctg aca cct ccc gaa gtg gac cac aag 1034 IleLeu Ala Lys Ala Asp Thr Leu Thr Pro Pro Glu Val Asp His Lys 290 295 300aaa cgc aaa atc cgg gag gag att gag cat ttt gga atc aag atc tat 1082 LysArg Lys Ile Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr 305 310 315caa ttc cca gac tgt gac tct gat gag gat gag gac ttc aaa ttg cag 1130 GlnPhe Pro Asp Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln 320 325 330gac caa gcc cta aag gaa agc atc cca ttt gca gta att ggc agc aac 1178 AspGln Ala Leu Lys Glu Ser Ile Pro Phe Ala Val Ile Gly Ser Asn 335 340 345350 act gta gta gag gcc aga ggg cgg cga gtt cgg ggt cga ctc tac ccc 1226Thr Val Val Glu Ala Arg Gly Arg Arg Val Arg Gly Arg Leu Tyr Pro 355 360365 tgg ggc atc gtg gaa gtg gaa aac cca ggg cac tgc gac ttt gtg aag 1274Trp Gly Ile Val Glu Val Glu Asn Pro Gly His Cys Asp Phe Val Lys 370 375380 ctg agg aca atg ctg gta cgt acc cac atg cag gac ctg aag gat gtg 1322Leu Arg Thr Met Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val 385 390395 aca cgg gag aca cat tat gag aac tac cgg gca cag tgc atc cag agc 1370Thr Arg Glu Thr His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser 400 405410 atg acc cgc ctg gtg gtg aag gaa cgg aat cgc aac aaa ctg act cgg 1418Met Thr Arg Leu Val Val Lys Glu Arg Asn Arg Asn Lys Leu Thr Arg 415 420425 430 gaa agt ggt acc gac ttc ccc atc cct gct gtc cca cca ggg aca gat1466 Glu Ser Gly Thr Asp Phe Pro Ile Pro Ala Val Pro Pro Gly Thr Asp 435440 445 cca gaa act gag aag ctt atc cga gag aaa gat gag gag ctg cgg cgg1514 Pro Glu Thr Glu Lys Leu Ile Arg Glu Lys Asp Glu Glu Leu Arg Arg 450455 460 atg cag gag atg cta cac aaa ata caa aaa cag atg aag gag aac tat1562 Met Gln Glu Met Leu His Lys Ile Gln Lys Gln Met Lys Glu Asn Tyr 465470 475 taactggctt tcagccctgg atatttaaat ctcctcctct tcttcctgtccatgccggcc 1622 cctcccagca ccagctctgc tcaggcccct tcagctactg ccacttcgcctaacatccct 1682 gctgactgcc cagagactca gaggaaataa agtttaataa atctgtaggtggc 1735 4 478 PRT Homo sapiens 4 Met Asp Arg Ser Leu Gly Trp Gln GlyAsn Ser Val Pro Glu Asp Arg 1 5 10 15 Thr Glu Ala Gly Ile Lys Arg PheLeu Glu Asp Thr Thr Asp Asp Gly 20 25 30 Glu Leu Ser Lys Phe Val Lys AspPhe Ser Gly Asn Ala Ser Cys His 35 40 45 Pro Pro Glu Ala Lys Thr Trp AlaSer Arg Pro Gln Val Pro Glu Pro 50 55 60 Arg Pro Gln Ala Pro Asp Leu TyrAsp Asp Asp Leu Glu Phe Arg Pro 65 70 75 80 Pro Ser Arg Pro Gln Ser SerAsp Asn Gln Gln Tyr Phe Cys Ala Pro 85 90 95 Ala Pro Leu Ser Pro Ser AlaArg Pro Arg Ser Pro Trp Gly Lys Leu 100 105 110 Asp Pro Tyr Asp Ser SerGlu Asp Asp Lys Glu Tyr Val Gly Phe Ala 115 120 125 Thr Leu Pro Asn GlnVal His Arg Lys Ser Val Lys Lys Gly Phe Asp 130 135 140 Phe Thr Leu MetVal Ala Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu 145 150 155 160 Val AsnSer Leu Phe Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu 165 170 175 GlyAla Glu Glu Arg Ile Met Gln Thr Val Glu Ile Thr Lys His Ala 180 185 190Val Asp Ile Glu Glu Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp 195 200205 Thr Pro Gly Phe Gly Asp Ala Val Asn Asn Thr Glu Cys Trp Lys Pro 210215 220 Val Ala Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe Arg Asp Glu225 230 235 240 Ser Gly Leu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val HisCys Cys 245 250 255 Leu Tyr Phe Ile Ser Pro Phe Gly His Gly Leu Arg ProLeu Asp Val 260 265 270 Glu Phe Met Lys Ala Leu His Gln Arg Val Asn IleVal Pro Ile Leu 275 280 285 Ala Lys Ala Asp Thr Leu Thr Pro Pro Glu ValAsp His Lys Lys Arg 290 295 300 Lys Ile Arg Glu Glu Ile Glu His Phe GlyIle Lys Ile Tyr Gln Phe 305 310 315 320 Pro Asp Cys Asp Ser Asp Glu AspGlu Asp Phe Lys Leu Gln Asp Gln 325 330 335 Ala Leu Lys Glu Ser Ile ProPhe Ala Val Ile Gly Ser Asn Thr Val 340 345 350 Val Glu Ala Arg Gly ArgArg Val Arg Gly Arg Leu Tyr Pro Trp Gly 355 360 365 Ile Val Glu Val GluAsn Pro Gly His Cys Asp Phe Val Lys Leu Arg 370 375 380 Thr Met Leu ValArg Thr His Met Gln Asp Leu Lys Asp Val Thr Arg 385 390 395 400 Glu ThrHis Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr 405 410 415 ArgLeu Val Val Lys Glu Arg Asn Arg Asn Lys Leu Thr Arg Glu Ser 420 425 430Gly Thr Asp Phe Pro Ile Pro Ala Val Pro Pro Gly Thr Asp Pro Glu 435 440445 Thr Glu Lys Leu Ile Arg Glu Lys Asp Glu Glu Leu Arg Arg Met Gln 450455 460 Glu Met Leu His Lys Ile Gln Lys Gln Met Lys Glu Asn Tyr 465 470475 5 24 DNA Artificial Sequence Description of Artificial SequencePrimer 5 ctgagcaagt tcgtgaagga tttc 24 6 23 DNA Artificial SequenceDescription of Artificial Sequence Primer 6 cagtcctctg acaaccagca gta 23

What is claimed is:
 1. A method for detecting human colorectal cancer, wherein the method comprises the steps of: bringing a specimen from a pathologic tissue of a patient into contact with an isolated polynucleotide having at least 97% homology with DNA encoding a bradeion protein which comprises an amino acid sequence 1-119 of SEQ ID NO:4 and which has the following properties: (i) it is a transmembranous protein; (ii) it has a transmembrane portion, an extracellular portion, and a cytoplasmic portion in its molecule as determined by a hydrophobicity analysis according to Kyte-Doolittle method; (iii) it is expressed in the human adult normal brain and heart, the expression level thereof in the heart being about 10% or lower of that in the brain, while it is not expressed in other adult normal organs of spleen, lung, liver, skeletal muscle, kidney and pancreas, and in fetal brain, lung, heart and kidney; (iv) it induces programmed cell death when over-expressed in a cultured human brain-derived undifferentiated nerve cell line; (v) it induces termination of cell division and aging when overexpressed in a cultured human brain-derived differentiated nerve cell; (vi) it is located in cytoplasm in the course of the induced cell death, and forms an intracellular aggregate when overexpressed; and (vii) it is expressed in a human colorectal cancer cell line or in a human malignant melanoma cell line, but not in leukemia, lymphoma and lung carcinoma; or a DNA fragment with at least 15 nucleotides encoding a fragment of said bradeion protein, or complements thereof; detecting the presence of a nucleic acid encoding said bradeion protein in said specimen; and determining whether there exists said cancer in said patient by using the presence of said nucleic acid as an indication.
 2. The method of claim 1, wherein said DNA is cDNA for bradeion protein, which is contained in a deposited plasmid, the accession number FERM BP-6922.
 3. The method of claim 1, wherein said DNA is cDNA, which encodes a bradeion protein having an amino acid sequence of SEQ ID NO:2 or
 4. 4. The method of claim 1, wherein said DNA is cDNA for bradeion protein having the nucleotide sequence 129-1943 of SEQ ID NO:1 or the nucleotide sequence 129-1562 of SEQ ID NO:
 3. 5. A method of claim 1, wherein said detection is conducted by hybridization or RT-PCR.
 6. The method of claim 1 wherein said DNA, DNA fragment or complements thereof is/are labeled.
 7. The method for detecting human colorectal cancer, wherein the method comprises the steps of: bringing a specimen from a pathologic tissue of a patient into contact with an antibody which is immunologically reactive with a bradeion protein which comprises an amino acid sequence having at least 97% homology to the amino acid sequence 1-119 of SEQ ID NO:4 and which has the following properties: (i) it is a transmembranous protein; (ii) it has a transmembrane portion, an extracellular portion, and a cytoplasmic portion in its molecule as determined by a hydrophobicity analysis according to Kyte-Doolittle method; (iii) it is expressed in the human adult normal brain and heart, the expression level thereof in the heart being about 10% or lower of that in the brain, while it is not expressed in other adult normal organs of spleen, lung, liver, skeletal muscle, kidney and pancreas, and in fetal brain, lung, heart and kidney; (iv) it induces programmed cell death when over-expressed in a cultured human brain-derived undifferentiated nerve cell line; (v) it induces termination of cell division and aging when overexpressed in a cultured human brain-derived differentiated nerve cell; (vi) it is located in cytoplasm in the course of the induced cell death, and forms an intracellular aggregate when overexpressed; and (vii) it is expressed in a human colorectal cancer cell line or in a human malignant melanoma cell line, but not in leukemia, lymphoma and lung carcinoma; detecting the presence of said bradeion protein in said specimen; and determining whether there exists said cancer in said patient by using the presence of said bradeion protein as an indication.
 8. The method of claim 7, wherein said antibody is elicited against a bradeion protein encoded by cDNA which is contained in a deposited plasmid, the accession number FERM BP-6922.
 9. The method of claim 7, wherein said antibody is elicited against a bradeion protein having an amino acid sequence of SEQ ID NO:2 or
 4. 10. A method of claim 7, wherein said detection is conducted by an immunoassay including enzymatic immunoassay, radioimmunoassay, or fluorescent antibody method.
 11. A method of claim 7, wherein said antibody is monoclonal.
 12. A method of claim 7, wherein said antibody is polyclonal.
 13. A method for detecting human malignant melanoma, wherein the method comprises the steps of: bringing a specimen from a pathologic tissue of a patient into contact with DNA encoding a bradeion protein, the DNA being contained in a deposited plasmid, the accession number FERM BP-6922, or the complement thereof; detecting the presence of a nucleic acid encoding said bradeion protein in said specimen; and determining whether there exists said cancer in said patient by using the presence of said nucleic acid as an indication.
 14. A method for detecting human malignant melanoma, wherein the method comprises the steps of: bringing a specimen from a pathologic tissue of a patient into contact with DNA encoding a bradeion protein having the nucleotide sequence 129-1943 of SEQ ID NO:1; detecting the presence of a nucleic acid encoding said bradeion protein in said specimen; and determining whether there exists said cancer in said patient by using the presence of said nucleic acid as an indication.
 15. A method of claim 13, wherein said detection is conducted by hybridization.
 16. A method of claim 13, wherein said DNA is labeled.
 17. A method of claim 14, wherein said detection is conducted by hybridization.
 18. A method of claim 14, wherein said DNA is labeled. 